Molecular pathogenesis of myeloproliferative neoplasms: Where do we stand in 2023?
Authors: Violaine Havelange, Stefan N. Constantinescu
Journal: American Journal of Hematology (2023)

Next-generation sequencing (NGS) in clinical practice and whole exome sequencing (WES) in research have in the last years accelerated the understanding of the molecular pathogenesis of myeloproliferative neoplasms (MPNs).1 The classical BCR-ABL1-negative MPNs arise from clonal proliferation of a single hematopoietic stem cell that will be responsible for the excessive production of mature myeloid cells.2 This heterogeneous group of diseases includes three subtypes: polycythemia vera (PV) involving predominantly the erythroid lineage, essential thrombocytemia (ET) with overproduction of megakaryocytes and platelets and primary myelofibrosis (PMF) characterized by fibrosis and splenomegaly. The acquisition of one gain-of-function “disease phenotypic driver” mutation (JAK2V617F, CALR, MPL) in a single hematopoietic stem cell can initiate and promote MPN disease without requiring additional cooperating mutation. As reported in this issue of the American Journal of Hematology, single-cell DNA sequencing studies reveal that MPNs appear to be oligoclonal rather than monoclonal (Figure 1).3 The three “disease phenotypic driver” mutations will lead to constitutive activation of the JAK/STAT signaling pathway, which is also found in triple-negative MPN patients. The time between the acquisition of the “disease phenotypic driver” mutation and the disease phenotype may be prolonged over decades.2 MPN “disease phenotypic driver” mutations (mainly JAK2V617F) can be detected in certain individuals with normal blood counts that exhibit clonal hematopoiesis of undetermined potential.4, 5 Mathematical models of prediction indicated that CALR mutations may be acquired later in life even if CALR-mutated MPNs develop on average a decade before JAK2V617F MPNs.6 However, JAK2V617F and CALR mutations were both detected in utero in some patients with a long latency before the development of the disease.7, 8 The JAK2V617F mutation is the most frequent disease phenotypic driver mutation detected in >95% of the PV and in 50%–60% of ET and PMF. JAK2 is a non-receptor tyrosine kinase, which is associated with the cytoplasmic domain of several cytokine receptors including MPL, EPOR, and G-CSF-R. JAK2V617 is a somatic G to T mutation at nucleotide 1849 in exon 14 of JAK2 resulting in the substitution of valine to phenylalanine at codon 617. This gain-of-function mutation is in the pseudokinase domain and will activate the kinase domain promoting dimerization of the mutated pseudokinase domain via a network of aromatic interactions, thus allowing dimerization and activation of the kinase domain and removing the negative regulation of the kinase domain.9-13 JAK2V617F will constitutively activate the three main myeloid homodimeric receptors (EPOR, G-CSFR, TPOR/MPL) and induce three phenotypes by activating mainly three signaling pathways STAT, PI3K/AKT/mTOR, and RAS/MPK. JAK2V617F can be heterozygous or become homozygous after mitotic recombination. Homozygous mutation is more frequent in PV patients. The variant allele frequency (VAF) in granulocytes is usually low in ET and higher in PV (> 50%) and close to 100% in post-PV or post-ET MF. The mutation of the calreticulin (CALR) is detected in 25%–30% of ET and in 20%–25% of PMF patients. Calreticulin is a chaperone protein that is involved in the quality control of the glycoproteins and in calcium metabolism. The most prevalent CALR mutations are mainly deletion of 52 bp (del52 – type 1) and insertion of 5 bp (ins5- type 2) in the exon 9, but more than 55 other mutations have been described that accomplish the same +1 frameshift. These frameshift mutations will result in a new C-terminus domain with positively charged hydrophobic residues lacking the KDEL endoplasmic reticulum (ER) retention signal.14 The differences between del52-like and ins5-like mutations, denoted as type 1 and type 2, also result from maintaining certain negatively charged residues from wild-type CALR in ins5/type 2 mutants, while del52/type 1 mutants eliminate all remaining negatively charged residues. The CALR mutant C-terminus domain forms a dimer and will bind to negatively charged patches on TPOR extracellular domain while the N-terminus lectin domain of CALR will bind to the N-glycans on TPOR extracellular domain Asn117; this bi-partite interaction induces TPOR dimerization, leading to a persistent JAK2 activation.14-18 The CALRm/TPOR complex will move to the cell surface or be secreted as a rogue cytokine, which will subsequently bind to mutated cells.17 CALR mutant will also impair calcium retention in the ER and activation of an ER stress response. The VAF of CALR mutation in the granulocytes in ET is usually high (40%). The myeloproliferative leukemia protein also known as thrombopoietin receptor (MPL/TPOR) mutations are rare and detected in 5% of PMF and 10% of ET patients. The main TPOR functions are the regulation of the platelet production and the maintenance of hematopoietic stem cells. MPL mutations are prevalent in the cytosolic juxta-membrane domain at W515 (W515L/W515K). These gain-of-function mutations result in a ligand-independent TPOR dimerization of the transmembrane domain, a constitutive activation of TPOR, an activation of JAK2 signaling pathways, and TPO-independent megakaryocytopoiesis.19-22 Mutations in MPL also have been described in the transmembrane domain (S505N).23 The transmembrane and juxtamembrane mutations induce dimerization of the TPOR transmembrane domain and unraveling (flexibility) of the region downstream the transmembrane domain, allowing JAK2 kinases to cross-phosphorylate and activate each other.24, 25 A fascinating aspect is that while both CALR and MPL/TPOR mutants function by persistent TPO-independent activation of TPOR itself, the phenotype, age, and evolution of the diseases are not identical, the MPL/TPOR mutated patients being closer to JAK2V617F patients than to CALR-mutated patients. It remains to be seen whether CALR mutant proteins activate TPOR in a different way or with different kinetics than mutations in TPOR itself, or whether other functions of mutant CALRs related to ER stress, NFkB, and Ca2+ would explain these differences.26 The “disease phenotypic driver” mutation influences the clinical phenotype, prognosis, and outcome. Young male patients with CALR-mutated ET present a lower hemoglobin level and white blood cell (WBC) count, but a higher platelet count compared with patients with JAK2 and MPL mutations in MPNs.27-29 CALR-mutated PMF patients have a lower WBC count and a higher platelet count than JAK2-mutated patients.30, 31 CALR-mutated PMF patients have a lower incidence of anemia requiring transfusion, thrombocytopenia, and leukocytosis compared with the JAK2V617F PMF patients.30, 31 CALR-mutated ET and PMF patients have a lower risk of thrombosis (divided by two) than JAK2-mutated patients.27, 31, 32 CALR-type 1 (del52-like) mutated patients seem to exhibit an increased risk of fibrotic transformation compared with JAK2-mutated patients.33 CALR-mutated PMF patients have an indolent clinical course and a better overall survival compared with JAK2 or MPL or TN PMF patients.30, 31, 34 The NGS data revealed additional somatic mutations called “clonal driver” mutations in some but not all MPN patients. These mutations may be associated with the phenotype and have a prognostic role in MPN patients. The most frequent MPN “clonal driver” mutations involved TET2, DNMT3A, ASXL1, EZH2, SRSF2, and SF3B1. These mutations are not MPN-specific. They are found in up to 20% of PV, up to 20% of ET, and up to 40% of PMF patients. Additional mutations usually occur after the “disease phenotypic driver” mutation and may accelerate the JAK2V617F-driven clonal expansion and contribute to disease progression and leukemic transformation. They can also occur as early events that facilitate the clonal emergence and the acquisition of the JAK2V617F mutation.35 PV and ET showed the same number of associated mutations, but MF patients present a higher number of mutations that are more heterogeneous.36 TET2 mutations are the most common and are detected in 10%–20% of classical MPN. DNMT3A mutations are detected in 5%–10% of MPN patients. TET2 and DNMT3A mutations are usually required for JAK2V617F-driven disease. CALR mutation is associated with fewer additional mutations. The order of the acquisition of the mutation influences the clinical phenotype and the prognosis.37 JAK2 first is associated with PV while TET2 first is associated with ET or MF. JAK2 first is associated with a higher risk of thrombosis. DNMT3A first is associated with ET. Tefferi et al. showed that spliceosome mutations are associated with a decrease in overall survival (SF3B1, SRSF2 in ET and SRSF2 in PV) and an increased risk of myelofibrosis evolution (U2AF1, SF3B1 in ET).38 Forty to sixty percent of MF patients harbor deleterious mutations. High molecular risk (HMR) mutations included EZH2, IDH1/2, SRSF2, ASXL1, and U2AF1. The presence of any HMR mutation in PMF patients confers a shorter overall survival. The detection and the number of HMR mutations are included in the MIPSS70+ version 2.0 and GIPPS prognostic scores. These prognostic scores are used for the decision of transplant. The myelodepletive–cytopenic phenotype of MF is characterized by a low JAK2V617F VAF frequency or a wild-type JAK2 and somatic mutations involving the spliceosome, epigenetic, and apoptosis pathways.39 Myeloid mutations including HMR and U2AF1 mutations are enriched in cytopenic MF patients, suggesting that this phenotype is caused less by JAK/STAT activating mutations and more by the evolution of an aggressive subclone with several clonal driver mutations.39, 40 The myelodepletive phenotype is more frequently observed in PMF patients and is associated with a shortened survival. Leukemic transformation of MPN holds still a dismal prognosis. The mutational profile of secondary AML (sAML) involved frequently mutated TP53, ASXL1, RUNX1, EZH2, IDH1/2, SRSF2, and SH2B3. These mutations are acquired at the time of leukemia transformation or are already detected at a very low allele frequency in chronic phase, where they can be missed by bulk DNA sequencing and are detected by single-cell DNA sequencing approaches such as Tapistry.3, 41 In some patients, JAK2V617F or CALR-mutated drivers are not detected in the leukemic blasts, suggesting that a secondary AML clone arises from a common mutated HSC before the acquisition of a disease driver like JAK2V617F or from another HSC.42 The molecular landscape of post-PV/ET AML is different from post-PMF. TP53 is the most frequent mutated gene in sAML and is mainly involved in post-PV or post-ET AML.43 Long-term transformation frequently involves TP53 mutation. TP53 mutations can be detected at a very low VAF in chronic-phase MPN patients. The leukemic transformation will require the loss of the second TP53 allele or another genetic event.44 Short-term transformation involves more complex additional mutations such as IDH1/2, DNMT3A, EZH2, U2AF1, and RUNX1 not detectable in the chronic phase.43 ASXL1 is more frequently involved in post-PMF AML.45 Single-cell sequencing advanced our understanding of the progression of MPNs to sAML. With respect to TP53-mutated sAML a recent study analyzed longitudinal cohorts of MPN and sAML patients by simultaneously single-cell sequencing DNA and RNA, allowing allelic resolution.46 Using analysis of antecedent TP53 heterozygous clones, a role for inflammation was established in the progression to sAML.46 In this journal, Calabresi et al. analyzed a cohort of MPNs that evolved to sAML by the single-cell DNA sequencing Tapestri approach combined with amplicon polyploidy. This analysis showed that MPNs may in fact be oligoclonal and that CNV in certain genes appear to be recurrent events in transformation. While the majority of sAML emerged from the driver disease clone maintaining, for example, JAK2V617F or CALR mutation, some (3/10) did not (Figure 1). A major question is whether in such cases the clone at the chronic stage triggers paracrine changes in another stem cell that becomes the course of AML. EZH2 emerged as the gene most frequently affected by single nucleotide and CNV, and this was validated at RNA seq and chromatin accessibility levels in one patient. Single-cell sequencing holds thus the potential to identify clonal complexity early on, when the future sAML clone is a minor clone at the chronic phase (Figure 1).3, 26, 41, 46, 47 Approaches allowing long-sequence determination (NanoPore technology) are being tested in many laboratories and it will be of great interest to compare the results obtained by various single-cell sequencing approaches. In conclusion, the deeper understanding of the molecular pathogenesis of MPN since 2005 has already allowed us not only to understand the physiopathology of the disease but also to develop diagnostic tools, prognostic scores, and new therapies. In the future, targeted therapies will be added to conventional therapies and adapted to each MPN molecular profile. The knowledge of the biochemistry of the mutations in the MPN cells allows the first development of immunotherapies and will participate in the progress of this promising therapy. Funding to VH is acknowledged from FNRS (Clinical Researcher), Salus Sanguinis, Fondation Roi Baudouin (Fonds Lucie et Monique Louette), Fonds Joseph Maisin, and Fondation Saint-Luc. Funding to SNC is acknowledged from Ludwig Institute for Cancer Research, FNRS Belgium, Fondation contre le cancer, Salus Sanguinis and Fondation “Les avions de Sébastien”, projects Action de recherché concertée (ARC) 16/21-073 and WELBIO F 44/8/5 – MCF/UIG – 10955, avenue Pasteur, 6, 1300 Wavre (Belgium). Stefan N. Constantinescu is a co-founder of MyeloPro Diagnostics and Research GmbH, Vienna.